Method of cell capture

ABSTRACT

Embodiments of methods and devices are disclosed for the manipulation (e.g., concentration, purification, capture, trapping, location, transfer) of analytes, e.g., biomolecules, with respect to analyte-containing solutions, using one or more electric fields.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/938,894 filed on Aug. 24, 2001, which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

This invention relates to methods and apparatus for the manipulation ofanalytes using electric fields.

BACKGROUND

In many chemical and biochemical methods and/or applications, it isoften desirable to at least partially concentrate or purify a sample oranalyte. For example, increased analyte concentration can enhancechemical reaction rates, rates of mass transfer, and/or detectability,etc. In addition, methods for controlling the location of at least aportion of a sample or analyte can be important in many chemical andbiochemical methods and/or applications.

SUMMARY

An aspect of the present invention is directed towards methods anddevices for the manipulation (e.g., concentration, purification,capture, trapping, location, transfer, and the like) of polarizableanalytes, such as polynucleotides, using one or more electric fields.

An embodiment of the present invention relates, in part, to a device andmethod for purification/concentration of an analyte away from aprocessor and/or analyzer (e.g., a capillary electrophoresis device, anoptical reader, a mass spectrometer, a chromatographic column, a PCRdevice, etc.). In an embodiment, a device and method of the presentinvention are useful in transferring a purified/concentrated sample to adesired location, such as to a sample-receiving region of such aprocessor and/or analyzer, and/or to a vessel such as a tube or a wellof a multi-well plate.

An embodiment of the invention makes use of the phenomenon that if amolecule or particle can be polarized, then it can be attracted orrepelled from a field gradient. Certain embodiments of the invention arebased in part on the discovery that when a polarizable analyte isattracted to a region having an alternating current electrical fieldgradient, the analyte can become trapped in a concentration zone formedby the alternating electric field. Various embodiments of the presentinvention, for example, contemplate polynucleotides, e.g., DNA or RNA,as polarizable molecules of interest, and utilize increasing fieldgradients to attract such molecules. One of the many applications of thepresent invention is to partition, at least in part, DNA from salts orfree dyes.

An aspect of the present invention provides an analyte-manipulationdevice for moving a polarizable analyte of interest, such as DNA or RNA,with respect to a sample holder (e.g., vial, tube, well, container,etc.) configured to hold such analyte. In an embodiment, the deviceincludes: at least two coextensive, elongated, electrically-conductivemembers disposed in spaced apart relation with respect to one another;with the members and the holder being adapted for relative movementbetween a first position wherein at least a portion of the members(e.g., lower end regions) is disposed within the holder and a secondposition wherein the members are disposed outside (e.g., spaced apart oraway from) of the holder; an AC power source adapted for electricalcommunication with the electrically-conductive members; wherein, withthe members and holder disposed at the first position, the AC powersource is operable in combination with the electrically-conductivemembers to establish an electrical field gradient within the holder,between the end regions.

Another aspect of the present invention provides an analyte-manipulationdevice for moving a charged analyte (e.g., biomolecule) of interest withrespect to a sample holder (e.g., vial, tube, well, container, etc.)configured to hold such analyte. In an embodiment, the device includes:at least two coextensive, elongated, electrically-conductive membersdisposed in spaced apart relation; with the members and the holder beingadapted for relative movement between a first position wherein at leasta portion of the members (e.g., lower end regions) is disposed withinthe holder and a second position wherein the members are disposedoutside of (e.g., spaced apart or away from) the holder; wherein atleast one of the members is a bubble-free electrode; a DC power sourceadapted for electrical communication with the electrically-conductivemembers; wherein, with the members and holder disposed at the firstposition, the DC power source is operable in combination with theelectrically-conductive members to establish an electrical potentialwithin the holder, between the members.

Numerous other advantages and features of the present invention willbecome apparent from the following detailed description of the inventionand the embodiments thereof, from the claims and from the accompanyingdrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are partially schematic, side and bottom views,respectively, of a device for manipulating analytes, according to theteachings of the present invention;

FIG. 2 is a side partial view of an embodiment of a device formanipulating analytes, according to the present invention;

FIG. 3 is a side partial view of an embodiment of a device formanipulating analytes, according to the present invention;

FIG. 4 is a side partial view of an embodiment of a device formanipulating analytes, according to the present invention;

FIG. 5A is a partially schematic view from one side of a device formanipulating analytes, according to an embodiment of the presentinvention;

FIG. 5B is a partial view from another side of the device of FIG. 5A;

FIG. 5C is a view from beneath the device of FIG. 5A;

FIGS. 5D and 5E are partially schematic views from a side and from oneend, respectively, of a device for manipulating analytes, according toan embodiment of the present invention;

FIG. 6 is a partially schematic side view of a device for manipulatinganalytes, according to an embodiment of the present invention;

FIG. 7 is a partially schematic side view of a device for manipulatinganalytes, according to an embodiment of the present invention;

FIG. 8 is a partially schematic side view of an embodiment of a devicefor manipulating analytes, according to the present invention;

FIGS. 9A and 9B are partially schematic side views of an embodiment of adevice for manipulating analytes, according to the present invention;and

FIG. 9C is a view of an end region of the device of FIGS. 9A and 9B,showing a gel-like electrolyte (slug) discharged therefrom.

Structural elements having similar or identical functions may have likereference numerals associated therewith. The appended drawingsillustrate only typical embodiments of this invention and are notlimiting of its scope, for the invention may admit to other equallyeffective embodiments.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Reference will now be made in detail to certain preferred embodiments ofthe present invention, examples of which are illustrated in theaccompanying drawings. While the invention will be described inconjunction with selected preferred embodiments, it will be understoodthat these embodiments are not intended to in any way limit the scope ofthe invention. On the contrary, the invention is intended to coveralternatives, modifications, and equivalents, which may be includedwithin the scope of the invention as determined by the appended claims.

As used herein, the term “purified” means that a material is removedfrom an original or starting state or environment. For example, amaterial is said to be “purified” when it is present in a particularcomposition in a higher concentration than exists as it is found in astarting sample. For example, where a starting sample comprises apolynucleoticle in a crude cell lysate, the polynucleotide can be saidnot to be purified, but the same polynucleoticle separated from some orall of the coexisting materials in the cell lysate is purified.

Generally, the present invention relates to methods and apparatus forthe manipulation (e.g., purification, concentration, capture, trapping,location, transfer, and the like) of analytes (e.g., bio-molecules)using one or more electric fields.

An aspect of the present invention makes use of field gradients toconcentrate and purify analytes in samples, including gradients formedby application of an AC field. Essentially any analyte that can bepolarized in an electric field, e.g., molecules or suspended particles,can be manipulated using generated field gradients. The inventors hereofhave found that polarizable analytes can be attracted to regions of highfield gradients, such as those generated near electrode edges or points.Further, in some cases, polarizable analytes can be repelled fromregions of high field gradient. Attraction to and/or repulsion fromregions of high field gradients are exploited herein as a means ofconcentrating polarizable particles, e.g., from a bulk solution.

In various embodiments, applied fields useful to attract or repulse apolarizable analyte are generated by way of an alternating current, orAC, power source. Preferably, the field is divergent at one or morepoints or regions in order to form field gradients to attract or repulsethe analytes.

Using attractive and/or repulsive concentration, a polarizable analytecan be located and concentrated relative to a bulk solution.Additionally, by selecting appropriate electric field parameters, apolarizable analyte can be purified relative to other species in a bulksolution that are less polarizable under the applied electric field.These less-polarizable analytes will feel a relatively smallerattraction or repulsion due to the applied field and will not be asreadily concentrated as will the more polarizable analyte. Thisdifference in selectivity for species based on attraction or repulsionto high field gradients can form the basis for concentration andpurification of one or more species from a complex mixture.

Aspects of the present invention are particularly useful in connectionwith analytes that have a dipole moment. The analyte may be charged oruncharged; it may have an overall net charge or be neutral. According tovarious embodiments, the analyte is present in a buffered electrolytesolution, e.g., an electrolyte solution having a low ionic strength.Exemplary analytes include nucleic acids, both single and doublestranded, proteins, carbohydrates, viruses, cells, organelles, organicpolymers, particles, and the like. In certain embodiments, a preferredanalyte for use with the present invention is single or double strandednucleic acid contained in an electrolyte solution.

In an embodiment of the invention, and with reference to FIG. 1, ananalyte-manipulation device, denoted generally as 10, is configured tocreate a field gradient; e.g., at a tip region, such is shown at 12. Thedevice 10 comprises a support 11 holding spaced-apart, co-extensive,elongate, electrically-conductive members, such as electrodes or wires14, 16. Members 14, 16 can be held in any suitable fashion. In certainpreferred embodiments, members 14, 16 are held in a fashion permittingthe members to move as a unit with support 11. For example, members 14,16 can be fixedly attached to support 11. In one embodiment, members 14,16 are firmly, yet removably, received within respective mounting holesformed through the support.

An elongate narrow gap region 18 is defined between confronting sideregions of members 14, 16. In an embodiment, with continued reference toFIG. 1, the dimension d of gap region 18 is within a range of from 20 μmand 1.50 mm. In a particular embodiment, the dimension d is betweenabout 100 μm and 0.75 mm. Members 14, 16 are disposed for electricalcommunication, via circuitry 19, with an alternating current (AC) powersupply 20 for providing a time-variant voltage difference therebetween.A concentration zone, indicated generally in the area denoted at 22, islocated along a submerged portion of the gap region 18, between members14, 16; and particularly near the tip region 12.

In use, the tip region 12 of the device 10 can be dipped into a samplecontaining one or more analytes of interest, such as the DNA-containingsample 23 held in tube or well 21 shown in FIG. 1A. For example, wherethe analyte of interest is a polynucleotide such as DNA, the DNA will bepreferentially attracted to an increasing field gradient established inthe gap region 18 between members 14 and 16; and particularly along theconcentration zone 22. In an embodiment, the gap region 18 is configuredto hold a defined volume of liquid, e.g., in a fashion similar to thatof a quill pen. The tip region 22 can then be moved to a receivingregion (e.g., a clean, empty well of a microtitre plate) were the DNAcan be deposited, e.g., upon discontinuing the AC field. The depositionof DNA into the receiving region can be expedited by applying a negativevoltage to members 14, 16 to drive the DNA off the tip region (due tothe repulsive effects of such negative voltage with respect to thenegatively charged phosphate groups of the DNA). FIG. 1A illustrates aDC power source 24 electrically connectable to (and disconnectable from)members 14, 16 by way of a switch 26 for such purpose.

Under circumstances where the receiving region (e.g., a vessel, such asa tube or well), whereat the DNA is deposited, contains a smaller amountof liquid the than the starting volume from which the DNA was taken, theDNA will thereby be concentrated. In addition, where the DNA is takenfrom a sample containing salts, the presently described device andmethod can assist in desalting the sample, since the tip region will notpreferentially attract salts (as salts are not readily polarized). Amongthe many advantages that can be realized by way of the presentteachings, the combination of these two events (concentration anddesalting) can result in an increase in the amount of analyte availablefor injection into a separation channel during a DNA analysis.

Optionally, the device 10 can further include a detector (not shown)positioned such that material located in the concentration zone 22 maybe detected. In addition, the device 10 can further optionally include acomputer (not shown) connected to one or both of power supplies 20, 24,and/or to the detector (not shown), for example, to control and monitorthe operation of the device and to manage the acquisition, analysis, andpresentation of data.

In an embodiment of the present invention, and with reference now toFIG. 2, device 10 includes elongate members 14, 16 with tip regions(generally at 12) that are potted in a non-conductive material, such asa bead of resin 30, or other non-conducting material (e.g., an epoxymaterial, or the like). This can be useful, for example, to assist inmaintaining a set (defined) spacing between the opposed tip regions.

In an embodiment of the invention, a nonconductive filament is wrappedaround one or more of the elongate members of the device. For example,FIG. 3 illustrates a non-conductive filament 34 wrapped in a spiral orhelical fashion around member 16. Suitable filament materials includenylon, Teflon, polyimide or other plastics, among others. The filamentcan be held in place against member 16, for example by tight wrappingand frictional forces and/or through the use of one or more adhesivesand/or fastening devices. The arrangement of FIG. 3 can help to maintaina fixed spacing between members 14 and 16, as well as increase theamount of fluid the device will hold. In addition, or as an alternative,the tip regions of members 14, 16 can be encapsulated with a porousmaterial (e.g., sponge-like), as at 36, to assist in holding fluid andkeeping the members 14, 16 spaced apart.

It is noted that the wires need not be rod-like or cylindrical. Avariety of geometries can be used to generate, tailor and even enhance(e.g., strengthen) the concentration of a field gradient. For example,surface features, such as points, sharp edges, corners, angles, bumps,protrusions, teeth, undulations, notches, indentations, waves, ripples,fins, or the like, can be provided along one or both of members 14, 16,e.g., in the vicinity of the tip regions. In one embodiment, one or bothelongated members are configured much in the fashion of a self-tappingscrew. Such features can be advantageous, for example, to form and/orenhance the field gradient. In an embodiment, illustrated in FIG. 4,sharp points or edges, indicated at 40, are formed along a majority ofthe length of each of members 14, 16 to each member's terminal end.

In an embodiment of the invention, an elongate non-conductive substrateis treated to provide conductive regions or paths therealong. Forexample, referring now to FIGS. 5A-5C, an elongate non-conductivesubstrate 111 bears separated coatings on opposite sides, as indicatedat 114 and 116. In one embodiment, electrically conductive coatings aresprayed or deposited onto a suitable non-conductive substrate, such as aporous ceramic or polymeric substrate. Any suitable electricallyconductive coating material can be used, e.g., metallic materials suchas copper or aluminum, or electrically conductive resins or paints. Inone embodiment a masking technique is employed to selectively depositconductive materials.

The present invention further contemplates arrangements wherein aplurality of analyte-manipulation devices as described herein are heldby a common support. FIGS. 5D-5E, for example, show a support 111 and aplurality of analyte-manipulation devices 110 depending therefrom. Eachof devices 110 is substantially as described above with respect to FIGS.5A-5C. As can be seen, for example, in FIG. 5D, this multiple-devicearrangement can be configured substantially in the fashion of a combhaving an upper elongate, substantially horizontally disposed bodyportion 115 and spaced apart elongate members 117, analogous to theteeth of a comb, depending therefrom. Body 115 and members 117 can beintegrally formed, or formed separately and attached to another oneanother by any suitable means (e.g., an adhesive or riveting). In thedepicted arrangement, body 115 and members 117 are comprised of anon-conductive material bearing coatings of conductive material onopposite major surfaces thereof to form electrical paths therealong. Theconductive coatings are disposed for electrical communication, viaappropriate circuitry 19, with an AC power source 20.

Another embodiment of the present invention includes a single elongateconductive member with a lower tip region and an AC power source. Areceiving region in connection with which the device is used, such aswell 21, can be electrically disposed (e.g., grounded) to provide areturn path. As shown for example in FIG. 6, well 21 includes aconductive surface 25 lining an inner, lower region thereof, which iselectrically coupled to AC power source 20. Surface 25 can be anysubstantially inert conductive material, such as a metallic layer. TheDNA contained in a fluidic sample 23 held in well 21 will bepreferentially attracted to the tip region 12 rather that the well 23because the small surface will cause field gradients in the vicinity ofthe tip region 12. It should be appreciated that surface features (e.g.,points, edges, etc.) or fluid capacity modifications, such as describedabove, can be employed in this embodiment.

According to an embodiment of the invention, an analyte-manipulationdevice of the present invention is configured for manual operation. Forexample, the support can be designed to be held in the hand of anoperator, e.g., in a fashion similar to that of a manually-operablepipette, and readily moved from place to place (e.g., from a samplevessel to a receiving vessel).

In another embodiment of the invention, one or more analyte-manipulationdevices, such as any of those described herein, are incorporated in anautomated workstation, e.g., such as described in U.S. Pat. No.6,132,582, entitled, “Sample handling system for a multi-channelcapillary electrophoresis device” and/or in U.S. Pat. No. 6,159,368,entitled, “Multi-well Microfiltration Apparatus” (both of which areincorporated herein by reference). For example, the support can beadapted for automated positioning (e.g., x-y-z motion) permitting thedevice to address various regions or components of a workstation.

As previously indicated, the electrodes used to effect the alternatingelectric field can have a shape which serves to form an electric fieldthat results in a defined concentration zone having desired dimensions.The spacing of the electrodes can be chosen so as to generate anelectric field having sufficient strength to trap a polar analyte ofinterest, but not so high as to cause excessive bubble formation at theelectrodes, e.g., see Washizu et al., IEEE Transactions on IndustryApplications, 30 (4): 835-843 (1994). In an embodiment, the spacing (seedimension d, in FIG. 1) between the electrodes is between about 20 μmand 2 mm (e.g., about 0.75-1.0 mm).

The alternating electric field used to trap a polarizable analyte can beany alternating electric field effective to manipulate, e.g., captureand concentrate, such an analyte. Generally, the alternating electricfield of the invention may be characterized by a time vs. field strengthprofile, a frequency, and a maximum field strength. The properties ofthe alternating electric field required to manipulate the analyte willdepend on a number of readily accessible experimental parametersincluding, for example, the magnitude of the dipole moment of a polaranalyte, the dielectric constant of the supporting medium, and, in thecase of an analyte having an induced dipole moment, the polarizabilityof the polar analyte or surrounding counter-ion microenvironment.

The time vs. field strength profile of the alternating electric fieldmay be sinusoidal, sawtooth, rectangular, superpositions of theforegoing, periodic or non-periodic, or any other profile capable ofbeing generated using a suitable function generator, e.g., a Model33120A 15 MHz Function/Arbitrary Waveform Generator from AgilentTechnologies. In an embodiment of the invention, the time vs. fieldstrength profile of the alternating electric field is rectangular. Inone embodiment, the time vs. field strength profile of the alternatingelectric field is such that the time-averaged integrated field strengthis zero where the average is taken over one complete cycle.

The frequency of the alternating electric field may be any frequencycapable of manipulating a portion of a polarizable analyte. For manyanalytes of practical importance, for example, the frequency of thealternating electric field may be between about 10 Hz and 100 megahertz(MHz). In an embodiment of the invention, the frequency of thealternating electric field is between 1 kilohertz (KHz) and 100 KHz.

While the maximum field strength of the alternating electric field maybe any field strength suitable to a particular application, in oneembodiment, the maximum field strength of the alternating electricfield, as measured by the peak field strength of the alternatingelectric field, is between about 100 V/cm and 20,000 V/cm; e.g., between1,000 V/cm and 10,000 V/cm. Preferably, the alternating electric fieldis spatially non-uniform.

In an embodiment of the invention, a trapped polar analyte is releasedfrom a concentration zone, for example, by reducing the trappingstrength of the alternating electric field. The trapping strength of thealternating electric field may be modulated by changing the frequency,field strength, or both. As previously indicated, another means foreffecting release of an analyte involves establishment of a DC fieldhaving a polarity opposite to that of a trapped polarized analyte.

In an embodiment, subsequent to a sample or analyte concentration stepaccording to the present invention, a further analytical step can beperformed. In one embodiment, after an analyte has been concentrated ina concentration zone, the analyte is directed into an analyticalseparation process, for example an electrophoretic or chromatographicseparation process. The concentration and localization methods of thepresent invention are particularly advantageous where the subsequentanalytical separation process is electrophoresis because thepre-separation concentration step can provide a concentrated and narrowinjection zone leading to both increased separation performance andenhanced detectability of the separated components.

In an embodiment of the present invention in which the analyte is anucleic acid, subsequent to or during the concentration of the analytenucleic acid in the concentration zone, the nucleic acid analyte issubjected to a nucleic acid hybridization reaction in which theconcentrated nucleic acid analyte is contacted with one or morecomplementary nucleic acids under conditions suitable forsequence-specific hybridization. In one embodiment, the complementarynucleic acids are bound to a solid support, e.g., an array ofsupport-bound nucleic acids including one or more potentiallycomplementary nucleic acids. The support-bound nucleic acids may besynthetic polynucleotide probes, cDNA molecules, or any other nucleicacid or nucleic acid analog capable of sequence-specific hybridization.Exemplary arrays of support-bound nucleic acids are described elsewhere,e.g., Singh-Gasson et al., Nature Biotechnology, 17: 974-978 (1999);Blanchard and Friend, Nature Biotechnology, 17: 953 (1999); Brown etal., U.S. Pat. No. 5,807,522; each of which is incorporated herein byreference. The pre-hybridization concentration step of the presentinvention may result in an increased rate of hybridization, the abilityto use a less concentrated sample, and/or an enhancement of thedetectability of the products of the hybridization reaction. While thisembodiment has been described in the context of nucleic acidhybridization, it will be apparent to one skilled in the art ofbiochemical instrumentation and analysis that this embodiment could beequally applied to other processes in which an analyte is contacted witha binding complement, e.g., antibody-antigen pairs, receptor-ligandpairs, biotin-avidin pairs, and the like.

Many bio-molecules in aqueous solution (e.g., suspension) areelectrically charged. As is well known, force acts between two objectswith charge, attractively between objects with opposite charges,repulsively between objects with similar charges. This property isexploited in certain embodiments of analyte-manipulation devices andmethods of the present invention.

Under some circumstances, concentration of charged analytes on ametallic electrode may be accompanied by the generation of bubbles. Insome situations, such bubbles may hinder or interfere with the desiredlocation of the analyte proximate or on the electrode. Accordingly, someembodiments of the present invention prefer the use of electrodes with areduced or eliminated tendency for bubble formation as compared to, say,similarly dimensioned and disposed platinum electrodes under likeconditions (referred to herein, collectively, as “bubble-free”electrodes).

In an embodiment of the present invention, by establishing a DC electriccurrent so as to establish a surface charge on the electrode that isopposite to the charge of the analyte, it is possible to attract theanalyte onto and/or alongside the surface of the electrode, therebyconcentrating the analyte. Or, where a gel-like electrode is employed,it is possible to attract the analyte to a region inside the electrode(e.g., via a process similar to electrokinetic injection). Uponreversing the polarity of the DC field so as to provide the electrodewith a charge like that of the concentrated analyte, the concentratedanalyte can be released or repelled from the electrode. One embodimentcontemplates transfer of the concentrated analyte by removing a portionof the electrode upon which the analyte has become situated.

Examples of solid electrodes employable in the present invention includePd, porous Pd, Ni(OH)₂, Ni(OH)₄, IrO₂. Also, ionic membranes, such asNafion, can be used. Examples of gel-like electrode arrangements includecapillary tubes or the like filed with gelatin, agarose, starch, or thelike. Examples of bubble-free electrodes and electrode materials, usefulin connection with the present invention, are described in co-filedpatent application Attorney Docket No. 5010-001 [Kilyk & Bowersox] and4573/4660 [Applied Biosystems], entitled, “Bubble-Free Electrophoresisand Electroosmotic Electrodes and Devices” naming as inventorsWoudenberg et al; which application is incorporated herein by referencein its entirety.

In an embodiment of the present invention, and with reference now toFIG. 7, an analyte-manipulation device 210 includes a first electrode214, e.g., a bubble-free electrode as discussed above, and a second(contra) electrode 216, each connected to a DC power source 224 by wayof appropriate circuitry 219. In use, electrodes 214, 216 can be placedin a solution, as shown, containing a charged analyte of interest, suchas solution 223 in tube or well 221. A DC current can be established sothat the charged analyte is electroplated on electrode 214, as indicatedat 259.

It should be noted that the particular positioning of the contraelectrode 216 is not critical. In one embodiment of the invention, thecontra electrode comprises part of a vessel (e.g., a cup, well or tube)in a fashion similar to that shown in FIG. 6. It is additionally notedthat it is not required that the contra electrode be bubble free (thoughit can be, if desired).

Referring now to FIG. 8, in an embodiment of an analyte-manipulationdevice according to the present invention, denoted generally by thereference numeral 310, a bubble-free electrode comprises a capillary ortube 361 (e.g., glass, fused silica, quartz) filled with an electrolyte363. One end 312 of the capillary 361 is covered by an ionic membrane367 (e.g., Nafion, or the like). The other end 369 is configured toreceive an electrically conductive element 373 (e.g., a Pt wire)therein, in contact with the electrolyte 363. In one embodiment, upperend 369 is vented, e.g., open to the atmosphere. An advantage of such anarrangement is that bubbles that might be generated during operation ofthe device, as indicated at 371, can be vented to the atmosphere. Anysuitable contra electrode arrangement (not shown) can be employed. Inoperation, device 310 can be placed in a solution containing a chargedanalyte of interest, and a DC current can be established so that thecharged analyte is attract to and collected on the ionic membrane 367.

As an alterative to the ionic membrane, or in addition thereto, ameltable plug that selectively allows passage of small ions but not ofmacromolecular analytes can placed in a region of end 312. For example,a meltable plug as disclosed in U.S. Pat. No. 5,593,559 (incorporatedherein by reference) can be placed at one or more regions inside thecapillary.

In an embodiment of the invention, referring now to FIGS. 9A-9C, acapillary or tube 461 is provided having one end 412 defining an orificeand a second end 469 disposed for communication with a pump 474, via aninterposed conduit 475. Any suitable pump can be employed, such as theillustrated piston-type pump having a piston configured for reciprocalmovement in the axial direction of a cylindrical reservoir. Pump 474 isadapted for pressure-filling the conduit and capillary with a gel-likeelectrolyte substance, denoted as 463. The electrolyte substance 463, inturn, is disposed for communication with a current source. Suchcommunication can be by any suitable means, and is preferably one thatis not associated with any substantial amount of bubble formation. Forexample, a bubble-free electrode (as at 473) or a porous membrane can beemployed. In an embodiment, the capillary or conduit containing theelectrolyte is provided with structure permitting electrical contactthrough ionic movement, as described in U.S. Re 35,102, incorporatedherein by reference. In one embodiment, GORE-TEX microporous PTFE tubingis utilized.

In operation, device 410 can be placed with the orifice at end 412submerged in a solution containing a charged analyte of interest (notshown). A DC current can be established so as to attract the chargedanalyte for collection on the gel. The concentrated analyte can then bemoved to a desired location and released by applying a reverse(repelling) voltage. The device can then be refreshed by operating thepump so as to force a portion of gel out through the open end of thecapillary (see FIG. 9B), which forced-out portion can then be removed(see FIG. 9C). It should be appreciated that, in some cases, it may bedesirable to dispense the analyte along with the gel. In an embodiment,the concentrated analyte is moved to a desired location and released bypumping the gel-like slug. In one such embodiment, for example, thegel-like slug is a gelatin material through which DNA does not migrate,and is melted once dispensed. Alternatively, an entangled polymer canact as a stacking media, being diluted once dispensed. Such dilution canchange the permeability of the polymer, thereby permitting it to act asa stacking media.

In an embodiment, electrically-conductive members of the device of theinvention (e.g., members 14, 16 of device 10 in FIG. 1; members 214, 216of device 210 in FIG. 7) are formed or treated so as to exhibit desiredwettability characteristics. For example, selected regions of members14, 16 (FIG. 1) can be provided with a surface that is hydrophilic,i.e., wettable. For example, selected regions of the surfaces of members14, 16 can be formed of a hydrophilic material and/or treated to exhibithydrophilic characteristics. In one embodiment, the surfaces havenative, bound or covalently attached charged groups.

Alternatively, or in addition, selected regions of members 14, 16 can beprovided with exterior surface regions that are hydrophobic, i.e., onethat causes aqueous medium deposited on the surface to bead. Forexample, selected regions of the exterior surfaces of members 14, 16 canbe formed of a hydrophobic material and/or treated to exhibithydrophobic characteristics. A variety of known hydrophobic polymers,such as polystyrene, polypropylene, and/or polyethylene, can be utilizedto obtain the desired hydrophobic properties. In addition, or as analternative, a variety of lubricants or other conventional hydrophobicfilms can be applied to a member's exterior surface.

In an embodiment, upper regions of the members 14, 16 are treated so asto be hydrophobic and, optionally, lower regions of the members aretreated so as to be hydrophilic.

It should be appreciated that instead of, or in addition to, having theelectrically-conductive members adapted for movement toward and/or awayfrom a sample holder, such as a tube or well; the sample holder can beadapted for movement.

All publications, patents, and patent applications mentioned herein arehereby incorporated by reference to the same extent as if eachindividual publication, patent, or patent application was specificallyand individually indicated to be incorporated by reference.

Those having ordinary skill in the art will readily understand that manymodifications are possible in the disclosed embodiments withoutdeparting from the teachings thereof. All such modifications areintended to be encompassed within the scope of the following claims.

1. A method comprising: generating an alternating electric field,between two co-extensive, elongated, electrically-conductive members;capturing polarizable cells in a sample, in the alternating electricfield, between the two co-extensive, elongated, electrically-conductivemembers, and removing the two co-extensive, elongated,electrically-conductive members with the polarizable cells capturedtherebetween, from the sample, while maintaining the alternatingelectric field.
 2. The method of claim 1, wherein the sample comprises acomplex mixture.
 3. The method of claim 1, wherein the alternatingelectric field has a time-averaged, integrated field strength that iszero when taken over one complete cycle.
 4. The method of claim 1,further comprising, contacting the sample with a binding componentbefore removing the two co-extensive, elongated, electrically-conductivemembers.
 5. The method of claim 1, further comprising: applying a firstdirect current electric field to the co-extensive, elongated,electrically-conductive members prior to generating the alternatingcurrent electric field.
 6. The method of claim 5 further comprisingapplying a second direct current electric field, oppositely chargedrelative to the first direct current electric field, to theco-extensive, elongated, electrically-conductive members after removingthe co-extensive, elongated, electrically-conductive members.
 7. Themethod of claim 1, wherein the polarizable cells are captured in a tipregion of the two co-extensive, elongated, electrically-conductivemembers.
 8. The method of claim 1, wherein the polarizable cells arepresent in a buffered electrolyte solution.
 9. The method of claim 1,wherein the removing comprises holding the device in an operator's hand.10. The method of claim 1, wherein the method further comprises, movingat least one of the co-extensive, elongated, electrically-conductivemembers between a cell pick-up position and a cell deposition position.11. The method of claim 1, wherein the capturing comprises concentratinga plurality of cells between the co-extensive, elongated,electrically-conductive members.
 12. The method of claim 1, wherein thecapturing comprises capturing a plurality of cells between a pluralityof pairs of co-extensive, elongated, electrically-conductive members.13. The method of claim 1, wherein the two co-extensive, elongated,electrically-conductive members are separated from one another by adistance of between about 20 micrometers and 2 millimeters.
 14. Themethod of claim 1, wherein the two co-extensive, elongated,electrically-conductive members are separated from one another by adistance of from 20 micrometers to 1.50 millimeters.
 15. The method ofclaim 1, wherein the two co-extensive, elongated,electrically-conductive members are separated from one another by adistance of between 100 micrometers and 0.75 millimeter.
 16. The methodof claim 1, wherein the two co-extensive, elongated,electrically-conductive members are separated from one another by adistance of between about 0.75 millimeter and 1.0 millimeter.